Methods of Using Substituted Tetracycline Compounds to Modulate RNA

ABSTRACT

A method for modulating RNA with tetracycline compounds is described.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/692,764 (now allowed), filed on Oct. 24, 2003, which claims priorityto U.S. Provisional Application Ser. No. 60/421,248, filed on Oct. 24,2002. The entire contents of the aforementioned prior applications arehereby incorporated by reference.

BACKGROUND

Molecules of RNA are transcribed from DNA. RNA molecules are relativelyshort compared to DNA molecules. RNA transcripts that direct thesynthesis of protein molecules are called messenger RNA (mRNA)molecules, while other RNA transcripts serve as transfer RNAs (tRNAs) orform the RNA components of ribosomes (rRNA) or smaller ribonucleoproteinparticles.

The amount of RNA made from a particular region of DNA is controlled bygene regulatory proteins that bind to specific sites on DNA close to thecoding sequence of a gene. In any cell at any given time, some genes areused to make RNA in very large quantities while other genes are nottranscribed at all. For an active gene thousands of RNA transcripts canbe made from the same DNA segment in each cell generation. Because eachmRNA molecule can be translated into many thousands of copies of apolypeptide chain, the information contained in a small region of DNAcan direct the synthesis of millions of copies of a specific protein.

In eukaryotes, a primary RNA transcript is synthesized; this transcriptcontains both introns and exons. Intron sequences are cut out and exonsequences on either side of an intron are joined together by RNAsplicing.

The translation of mRNA into protein depends on a set of small RNAmolecules known as transfer RNAs (tRNAs), each about 80 nucleotides inlength. A tRNA molecule has a folded three-dimensional conformation thatis held together in part by noncovalent base-pairing interactions likethose that hold together the two strands of the DNA helix. In thesingle-stranded tRNA molecule, however, the complementary base pairsform between nucleotide residues in the same chain, which causes thetRNA molecule to fold up in a unique way.

The codon recognition process by which genetic information istransferred from mRNA via tRNA to protein depends on the same type ofbase-pair interactions that mediate the transfer of genetic informationfrom DNA to DNA and from DNA to RNA. The mechanics of ordering the tRNAmolecules on the mRNA require a ribosome. Each ribosome is a largeprotein-synthesizing machine on which tRNA molecules position themselvesso as to read the genetic message encoded in an mRNA molecule. Theribosome first finds a specific start site on the mRNA that sets thereading frame and determines the amino-terminal end of the protein.Then, as the ribosome moves along the mRNA molecule, it translates thenucleotide sequence into an amino acid sequence one codon at a time,using tRNA molecules to add amino acids to the growing end of thepolypeptide chain. When a ribosome reaches the end of the message, bothit and the freshly made carboxyl end of the protein are released fromthe 3′ end of the mRNA molecule into the cytoplasm.

Although most tRNAs are initially synthesized as a larger precursor RNA,an RNA molecule has been shown to play the major catalytic role in anRNA-protein complex that recognizes these precursors and cleaves them atspecific sites. A catalytic RNA sequence also plays an important part inthe life cycle of many plant viroids. Most remarkably, ribosomes are nowsuspected to function largely by RNA-based catalysis, with the ribosomalproteins playing a supporting role to the ribosomal RNAs (rRNAs), whichmake up more than half the mass of the ribosome. The large rRNA byitself; for example, has peptidyl transferase activity and catalyzes theformation of new peptide bonds.

The development of compositions and methods for modulation of RNA wouldbe of great benefit in modulating numerous cellular processes and in thetreatment of disorders.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains at least in part, to methodsfor modulating RNA. The method includes contacting an RNA molecule or acellular component with a substituted tetracycline, such that modulationof RNA occurs.

In yet another embodiment, the invention includes a method for treatinga subject for a disorder treatable by modulation of RNA or by modulationof RNA in combination with a second agent (DTMR). The method includesadministering to the subject an effective amount of a tetracyclinecompound, or with a tetracycline compound in combination with a secondagent such that the DTMR is treated. In further embodiments, theeffective amount is effective to modulate translation, the half-life,message translocation, the binding of proteins, or splicing of thesubject's RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting variations of iNOS mRNA, protein, andnitrite levels in LPS stimulated mouse macrophages.

DETAILED DESCRIPTION OF THE INVENTION I. Methods for Modulating RNA

In one embodiment, the invention pertains at least in part, to methodsfor modulating RNA. The method includes contacting an RNA molecule, or acellular component, with a tetracycline compound, such that modulationof RNA occurs. In certain embodiments, the RNA molecule is locatedwithin a subject.

The term “modulate,” “modulating” or “modulation” includes increasing,decreasing, or otherwise changing the level, amount, function,structure, and/or activity of a particular molecule of RNA.

The term “modulating RNA” or “modulation of RNA” includes modulation ofall functions, structures, amounts, and/or activities of RNA which canbe modulated by substituted tetracycline compounds of the invention.Modulation of RNA includes, for example, modulation of transcription,translation, translocation, catalysis, secondary structure, splicing,stability, etc. The term also includes modulations of the half-life ofRNA. RNA can be modulated within an organism, cell, intracellular space,nucleus and/or other cellular compartment.

In one embodiment, a specific RNA molecule can be modulated, e.g., aspecific type of RNA (such as mRNA or rRNA) and/or an mRNA specifying aparticular protein can be modulated, while other mRNA molecules are notaffected. In another embodiment, RNA molecules in general, e.g., severaldifferent types of RNA can be modulated and/or a plurality of mRNAmolecules specifying different proteins, can be modulated according tothe invention.

Modulation of RNA can occur directly, e.g., by modulation of RNA itself,for example by binding of tetracycline to the RNA (for example to alterits secondary structure) or indirectly, e.g., by binding of a moleculeto a component of a cell, e.g., a protein with which the RNA interacts.For example, the tetracycline compound may interact with a particularprotein necessary for the synthesis of an RNA molecule or with a proteinmolecule with which the RNA molecule interacts (e.g., a ribosomalprotein), and thus modulate the RNA without directly binding to the RNAitself.

Examples of RNA molecules which may be modulated using the methods ofthe invention include, but are not limited to, hnRNA, mRNA, tRNA,ribosomal RNA, nuclear RNA, snRNA, and small RNA aptamers.

The RNA may also be e.g., RNA from a prokaryotic cell, a eukaryotic cellor may be viral RNA.

The term “cellular component” includes cells (in vivo and in vitro),cellular organelles (e.g., ribosomes, nuclei, mitochondria,chloroplasts, etc.), cytoplasm, etc. In a further embodiment, the cellsare located within a subject. In another embodiment, the cells are invitro. In another further embodiment, the cellular component comprisesRNA. In a further embodiment, the cellular component is a cell which isassociated with (e.g., derived from a subject having or present in asubject having) a particular disorder treatable by modulation of RNA orby modulation of RNA in combination with a second agent (DTMR). Forexample, when the DTMR is a tumor, the cellular component may be a cellof the tumor.

RNA modulation can occur via a variety of different mechanisms.Exemplary mechanisms are listed below.

In one embodiment, RNA is modulated by direct interaction with atetracycline molecule (e.g., Berens. 2001. Tetracyclines in Biology,Chemistry and Medicine ed. By M. Nelson, W. Hillen and R. A. Greenwald,pp 177-196). Preferably, “modulation of RNA” as used herein excludesinteraction of a tetracycline molecule with the 30S ribosomal subunit ofa bacterial cell. In another preferred embodiment, binding to 16S RNAand the proteins S4, S7, S9, and S17 are preferentially excluded fromthe term “modulation of RNA”.

In one embodiment, the RNA is modulated by altering RNA transcription.For example, tetracycline compound may inhibit or decrease thetranscription of an mRNA. In another embodiment, a tetracycline compoundmay inhibit transcription.

Levels of transcription can be measured in the presence and the absenceof a tetracycline compound using techniques that are known in the art.Transcription of a specific gene can be measured or genome-widetranscription (transcription of many genes) can be detected. Forexample, in one embodiment, transcription levels can be detected byperforming nascent-chain run-on analysis. This technique is known in theart and requires using P³² labeled nucleotides; genes with hightranscription levels can be detected by intensity. In another example,transcription of a reporter gene, e.g., luciferase which is easilydetectable, can be operably linked to a gene of interest. Detection oflight will indicate transcription of the gene of interest. Otherexemplary methods for measuring transcription include Northern blots andin situ hybridization. Detection of transcription levels of more thanone gene can be performed using, e.g., microarrays (e.g., cDNA orsynthetic oligonucleotide arrays) or PCR.

In one embodiment, the RNA is modulated by altering RNA translation. Forexample, tetracycline compound may inhibit or decrease the translationof a mRNA. In one embodiment, a tetracycline compound may inhibit RNAtranslation by inhibiting its initiation. In another embodiment, atetracycline compound may inhibit translation by altering the point atwhich translation terminates. For example, in one embodiment atetracycline compound can cause a ribosome to skip a termination codonand continue translation.

In one embodiment, the level of a specific protein translated from mRNAcan be measured using standard techniques. For example, in vitro or insitu analysis of enzyme activity can be measured, if the protein is anenzyme. In vitro analysis can include activity in bulk protein extracts,or after electrophoresis to partially separate the enzyme from otherproteins. In another example, in vitro or in situ analysis can beperformed using immunochemical methods, i.e., employing a labeledantibody specific for the protein. Quantification/visualization of theantibody can the be performed. Western blots can be performed afterelectrophoresis or cellular extracts or components can be assayeddirectly, e.g., by ELISA or immunoprecipitation. If the protein issufficiently abundant, it can also be directly visualized after 1D or 2Delectrophoresis if it can be separated sufficiently from other proteinsby this method.

In one embodiment, the level of mRNA specifying a particular protein canbe measured. In another embodiment, the level of total mRNA can bemeasured. Such measurements can be made using techniques describedherein or other techniques known in the art.

In another embodiment, the half-life of RNA is modulated by contactingthe cellular component with the tetracycline compound. For example, inone embodiment, the half-life of mRNA is increased. In one embodiment, atetracycline compound of the invention increases the binding of RNA to aribosome, thereby increasing the stability of the RNA (Wei andBechhofer. 2002. J. of Bacteriology 184: 889). In another embodiment,the half-life of mRNA is decreased. In a further embodiment, thetetracycline compound is not tetracycline or otherwise described in Weiand Bechhofer. 2002. J. of Bacteriology 184:889.

For example, in one embodiment, a tetracycline molecule of the inventionincreases the degradation of a specific mRNA molecule. For example, thehalf-life of mRNA specifying a protein such as iNOS (Amin et al. 1997.FEBS Letters 410:259) can be measured. In a further embodiment, thetetracycline compound is not doxycycline, minocycline, or a tetracyclinecompound described in Amin et al. 1997. FEBS Letters 410:259.

In one embodiment, the half-life of RNA can be measured using in vitronuclear run-on transcription assays known in the art. Nuclei can beisolated from cells and incubated in vitro with radioactive precursorsunder conditions where nascent RNA pol II will continue elongation offof the native gene, but will not initiate transcription. The fraction oftotal incorporated radioactivity in a specific transcript can bemeasured and a degradation rate constant can be generated. In anotherembodiment, a kinetic analysis can be performed. For example,radioactive precursors can be provided and, over time, amounts ofradioactivity (specific activity) in a particular mRNA can be measuredby hybridization with unlabeled cloned DNA. The concentration of mRNAcan be followed over time using this method.

In another embodiment, the Kd can be independently assayed by performinga pulse-chase experiment where radioactive precursor is chased out ofthe cell, and then the decline in radioactivity of mRNA molecules madeduring the pulse is followed. In yet another example, synthesis and/ordegradation rates can be estimated using transcription reporters.

In another embodiment, the RNA can be modulated by modulating thetranslocation of the RNA. For example, in one embodiment, a tetracyclinemolecule may interfere with the translocation of an RNA molecule to orfrom the nucleus of a cell.

Translocation of RNA to the nucleus can be measured, e.g., by nucleartranslocation assays in which the emission of two or morefluorescently-labeled species is detected simultaneously. For example,the cell nucleus can be labeled with a known fluorophore specific forDNA, such as Hoechst 33342. The RNA can be directly or indirectlylabeled, e.g., fluorescently-labeled antibody specific for RNA. Theamount of RNA that translocates to or from the nucleus can be determinedby determining the amount of a first fluorescently-labeled species,i.e., the nucleus, that is distributed in a correlated oranti-correlated manner with respect to a second fluorescently-labeledspecies, i.e., the RNA as described in U.S. Pat. No. 6,400,487, thecontents of which are hereby incorporated by reference.

Modulation of RNA also includes modulation of the processing of aparticular RNA molecule by splicing. The tetracycline compound mayaffect the arrangement, or the inclusion, or the exclusion of sectionsof the RNA by affecting the mechanisms governing splicing. For example,in the case of mRNAs, the tetracycline compound may, for example,promote the inclusion of a particular exon, or promote the exclusion ofa particular exon, or cause a particular exon size to become altered,for example, by inclusion of a sequence at the 5′ or the 3′ ends of theexon. The tetracycline compound may promote the inclusion or theexclusion of an exon containing, for example, a premature stop codon.The tetracycline compound may modulate splicing by, for example,activating cryptic splice sites, or silencing consensus splice sites, orsilencing exonic or intronic splicing enhancers (ESEs or ISEs) or bysilencing exonic or inronic splicing silencers (ESSs or ISSs), oraltering the binding orf a component of the splicing machinery to theRNA, or by affecting the intermolecular interactions between componentsof the splicing machincery. Examples of RNA splicing are discussed inStoss et al. (2000), Gene Ther. Mol. Biol. 5:9-30; Liu et al. (1994) J.Euk. Microbiol. 41:31; Hertweck et al., 2002. Eur. J. Biochem 269:175.

In another embodiment, the amount of spliced mRNA specifying aparticular protein can be measured. In another embodiment, the effect ofa tetracycline compound on splicing of RNA can be measured, e.g., usingstandard assays such as β globin splicing assays (Hertweck et al. 2002.Eur. J. Biochem. 269:175). In one embodiment, a particular form of RNA(e.g., an mRNA molecule comprising a particular exon) can be measured ina cell. In a further embodiment, the tetracycline compound is nottetracycline, chlortetracycline, or other tetracycline compoundsdescribed in Hertweck et al. 2002. Eur. J. Biochem. 269:175 or Liu etal. 1994. J. Euk. Microbiol. 41(1):31.

Various spliced forms of mRNA can be detected in a cell using techniquesknown in the art. For example, in one embodiment, PCR can be performedusing primer sets that specifically amplify the products to be detected(see, e.g., Lim and Hertel. 2001 J. Biol. Chem 276:45476). In anotherembodiment, a reporter cell line is used to detect changes in RNAsplicing. For example, a cell line such as the 654 EGFP reporter cellline (which comprises a C to T mutation at nucleotide 654 of the humanβ-globin intron 2 (see, e.g., Sazani et al. 2001. Nucleic Acids Research29:3965). Treatment of these cells with an agent that modulates RNAsplicing can restore proper splicing and translation of EGFP, therebyproviding a rapid and positive readout for identification of suchagents.

In another embodiment, the RNA is modulated by altering the interactionsof proteins with the RNA molecule. Examples of proteins which interactwith RNA include hnRNP proteins, snRNP proteins, ribosomal proteins,endonucleases, and other enzymes. The substituted tetracycline compoundmay either promote or inhibit the interactions of particular proteinswith RNA. In certain embodiments, the interaction of RNA with anothernucleic acid molecule may also be modulated by the interaction of thetetracycline compound.

The ability of the tetracycline compound to modulate binding of an RNAmolecule to one or more proteins can also be determined. Determining theability of the test compound to binding can be accomplished, forexample, by coupling the RNA molecule or the protein(s) with aradioisotope or enzymatic label such that binding of the RNA to theprotein can be determined by detecting the labeled molecule in acomplex. For example, RNA or protein can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. Alternatively, if the protein withwhich the RNA interacts is an enzyme, it can be detected withoutlabeling.

In one embodiment, the amount of binding of RNA to the protein targetmolecule in the presence of the tetracycline compound is greater thanthe amount of binding of RNA to the target molecule in the absence ofthe tetracycline compound, in which case the tetracycline compound isidentified as a compound that enhances binding of RNA. In anotherembodiment, the amount of binding of the RNA to the target molecule inthe presence of the tetracycline compound is less than the amount ofbinding of the RNA to the target molecule in the absence of thetetracycline compound, in which case the tetracycline compound isidentified as a compound that inhibits the binding of RNA to protein.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either RNA or a targetprotein molecule, for example, to facilitate separation of complexedfrom uncomplexed forms of one or both of the molecules, or toaccommodate automation of the assay. Binding of a tetracycline compoundto an RNA molecule, or interaction of an RNA molecule with a proteinmolecule in the presence and absence of a test compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix.

In one embodiment, RNA modulation can be detected in a cell by measuringthe effect of a tetracycline compound on the amount or activity of oneor more proteins in a cell. Preferably the protein is one associatedwith a particular disorder in a subject, i.e., is a therapeuticallyrelevant protein.

In another aspect, the invention pertains to a method for treating asubject for a disorder treatable by modulation RNA or by modulation ofRNA in combination with a second agent (DTMR). The method includesadministering to the subject an effective amount of a substitutedtetracycline compound or an effective amount of a substitutedtetracycline compound and a second agent (e.g., a chemotherapeuticagent) such that the DTMR is treated.

The term “disorders treatable by modulation of RNA” or “DTMR” includesviral, neurodegenerative and other disorders which are caused or relatedto RNA function, structure, amounts and/or other activities of RNA whichare lower or higher than desired and those disorders treatable bycompounds described herein. Examples of DTMR include viral disorders(e.g., retroviral disorders (e.g., HIV, etc.), disorders caused by humanrhinovirus RNA and proteins, VEE virus, Venezuelan equine encephalitisvirus, eastern X disease, West Nile virus, bacterial spot of peach,camelpox virus, potato leafroll virus, stubborn disease and infectiousvariegations of citrus seedlings, viral protein synthesis in Escherichiacoli infected with coliphage MS2, yellow viruses, citrus greeningdisease, ratoon stunting disease, European yellows of plants, inclusionconjunctivitis virus, meningopneumonitis virus, trachoma virus, hogplague virus, ornithosis virus, influenza virus, rabies virus, viralabortion in ungulates, pneumonitis, and cancer.

Other exemplary DTMRs include disorders caused by, or associated withsplicing. For example, some disorders associated with defects inpre-mRNA processing result from a loss of function due to mutations inregulatory elements of a gene. Examples of such mutations are describedin Krawczak et al. (1992) Hum. Genet, 90:41-54; and Nakai et al. (1994)Gene 14:171-177. Other DTMR include disorders which have been attributedto a change in trans-acting factors. Examples of DTMRs which areassociated with splicing include those described in Philips et al.(2000), Cell. Mol. Life Sci., 57:235-249), as well as, FTDP-17(frontotemporal dementia with parkinsonism) and β-thalassemia.

Certain DTMRs associated with splicing include those which are generatedby point mutations that either destroy splice-sites or generate newcryptic sites in the vicinity of normally used exons. Examples of suchDTMRs include cystic fibrosis (Friedman et al. (1999) J. Biol. Chem.274:36193-36199), muscular dystrophy (Wilton et al. (1999) Neuromuscul.Disord. 9:330-338), and eosinophilic diseases (Karras et al., (2000)Mol. Pharamcol. 58:380-387).

Other DTMRs include cancers which may change splicing patterns duringcancer formation and progression. Example of such cancers include, butare not limited to leukemia, colon/rectal cancer, myeloid leukemia,breast cancer, gastric carcinomas, acute leukemia, multiple myeloma,myeloid cell leukemia, lung cancer, prostate cancer, etc. Addition DTMRsassociated with splicing are discussed in Stoss et al., (2000), GeneTher. Mol. Biol. 5:9-30).

Another example of a DTMR is a cancer in which treatment of the cancercells with a tetracycline compound results in the modulation of RNA,where the modulation of RNA increases the susceptability of the cell toa second agent, e.g., a chemotherapeutic agent. Such DTMRs can betreated using a combination of the tetracycline compound and achemotherapeutic agent. Exemplary cancers include those in which thetetracycline compound modulates the form of BCL expressed by the cells.

Other DTMRs include disorders wherein particular ribozymes are presentin aberrant quantities. Examples include breast cancer, hepatitis Cvirus (HCV), liver cirrhosis, and heptacellular carcinoma.

In a further embodiment, the tetracycline compounds for treating cancerdo not include, for example, the tetracycline compounds described inU.S. Pat. Nos. 6,100,248; 5,843,925; 5,837,696; 5,668,122; WO 98/31224;US 20020045603; WO 99/49871; WO 01/87823; WO 00/28983; U.S. Pat. No.5,574,026; incorporated herein by reference in their entirety.

Other DTMRs include, but are not limited to, asthma, arthritis, anemia,Alzheimer's, Huntington's disease, aortic aneurysm, diabetes, ischemia,hyperlipidemia, and obesity.

In an embodiment, when the DTMR is an aortic aneurysm, the tetracyclinecompound is not doxycycline. In another embodiment, when the DTMR isHuntington's disease, the tetracycline compound is not minocycline. Inanother embodiment, when the DTMR is cerebral ischemia, the tetracyclinecompound is not tetracycline. In other embodiments, when the DTMR isasthma, the tetracycline compound is not minocycline or doxycycline.

In other embodiments, the DTMRs of the invention do not include aorticaneurysm, Huntington's disease, asthma or cerebral ischemia.

The term “subject” with reference to treatment includes humans and otherorganisms and viruses which have RNA such as plants, animals (e.g.,mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits,squirrels, bears, primates (e.g., chimpanzees and gorillas)).

The language “effective amount” of the tetracycline compound is thatamount necessary or sufficient to treat or prevent a DTMR or modulateRNA in a subject. The effective amount can vary depending on suchfactors as the size and weight of the subject, the particular DTMR, orthe particular tetracycline compound. For example, the choice of thetetracycline compound can affect what constitutes an “effective amount”.One of ordinary skill in the art would be able to study theaforementioned factors and make the determination regarding theeffective amount of the tetracycline compound without undueexperimentation.

The regimen of administration can affect what constitutes an effectiveamount. The tetracycline compound can be administered to the subjecteither prior to or after the onset of a disease which is treatable.Further, several divided dosages, as well as staggered dosages, can beadministered daily or sequentially, or the dose can be continuouslyinfused, orally administered, administered by inhalation, or can be abolus injection. Further, the dosages of the tetracycline compound(s)can be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

The term “treated,” “treating” or “treatment” includes therapeuticand/or prophylactic treatment. The treatment includes the diminishmentor alleviation of at least one symptom associated or caused by the DTMR.For example, treatment can be diminishment of one or several symptoms ofa disorder or complete eradication of the DTMR.

In another aspect, the invention pertains to methods for identifyingtetracycline compounds for treating DTMR, comprising: contacting acellular component with a tetracycline compound; measuring the abilityof the tetracycline compound to modulate RNA, to thereby identify atetracycline compound for treating DTMR, either alone or in combinationwith a second agent.

In one embodiment, the ability of the compound to modulate RNAtranslation is measured. In another embodiment, the ability of thecompound to modulate the half-life of RNA is measured. In anotherembodiment, the ability of the compound to modulate translocation of RNAis measured. In another embodiment, the ability of the compound tomodulate the interaction of RNA with proteins is measured. In anotherembodiment, modulation of RNA splicing is measured. Modulation of RNAcan be detected using any of the methods described herein or other artrecognized methods.

II. Substituted Tetracycline Compounds

In one embodiment, the tetracycline compound is a substitutedtetracycline compound.

The term “tetracycline compound” includes substituted tetracyclinecompounds and compounds with a similar ring structure to tetracycline,including minocycline, doxycycline, tetracycline, chlortetracycline,oxytetracycline, demeclocycline, methacycline, sancycline, chelocardin,rolitetracycline, lymecycline, apicycline; clomocycline, guamecycline,meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline,penimocycline, etc. Other derivatives and analogues comprising a similarfour ring structure are also included (See Rogalski, “ChemicalModifications of Tetracyclines,” the entire contents of which are herebyincorporated herein by reference). Table 1 depicts tetracycline andseveral known other tetracycline derivatives.

TABLE 1

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12α-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6α-deoxy-4-dedimethylaminotetracycline;4-dedimethylamino-12α-deoxyanhydrotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a C1-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a,6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a,11a dehydro tetracyclines; 11aC1-6,12 hemiketal tetracyclines; 11a C1-6-methylene tetracyclines; 6,13diol tetracyclines; 6-benzylthiomethylene tetracyclines;7,11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines;6-fluoro(α)-6-demethyl-6-deoxy tetracyclines;6-fluoro(β)-6-demethyl-6-deoxy tetracyclines; 6-α acetoxy-6-demethyltetracyclines; 6-β acetoxy-6-demethyl tetracyclines;7,13-epithiotetracyclines; oxytetracyclines; pyrazolotetracyclines; 11ahalogens of tetracyclines; 12a formyl and other esters of tetracyclines;5,12a esters of tetracyclines; 10,12a-diesters of tetracyclines;isotetracycline; 12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxytetracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines. In certainembodiments, the term tetracycline compound does not include7-chlorotetracycline, minocycline, doxycycline, or tetracycline.

The term “tetracycline compounds” includes substituted tetracyclinecompounds as defined below, and as described in the specification. Thetetracycline compounds may or may not have antibacterial orantiinfective activity. In certain embodiments of the invention, thetetracycline compound has antiinfective, antiinflammatory and/orantibacterial activity. In other embodiments of the invention, thetetracycline compound does not have significant antiinfective,antiinflammatory or antibacterial therapeutic activity.

Examples of substituted tetracycline compounds include compoundsdescribed in U.S. Pat. Nos. 6,165,999; 5,834,450; 5,886,175; 5,567,697;5,567,692; 5,530,557; 5,512,553; 5,430,162 each of which is incorporatedherein by reference in its entirety. Other examples of substitutedtetracycline compounds include those described in, for example, WO99/37307, WO 02/12170, WO 02/04407, WO 02/04406, WO 02/04404, WO01/98260, WO 01/98259, WO 01/98236, WO 01/87824, WO 01/74761, WO01/52858, WO 01/19784, WO 84/01895, U.S. Ser. No. 60/367,050, U.S. Ser.No. 09/895,797, U.S. Ser. No. 60/305,546, U.S. Ser. No. 60/346,930, U.S.Ser. No. 60/346,929, U.S. Ser. No. 60/347,065, U.S. Ser. No. 60/346,956,U.S. Ser. No. 60/367,049, U.S. Ser. No. 10/097,095, U.S. Ser. No.10/097,135, U.S. Ser. No. 60/362,654, U.S. Ser. No. 60/367,045, U.S.Ser. No. 60/366,915, U.S. Ser. No. 60/367,048, and 10/196,010. Otherexamples of substituted tetracycline compounds are described in EP0582810 B1; EP 0536 515B1; EP 0582 789B1; EP 0582 829B1; EP 0582788B1;U.S. Pat. No. 5,530,117; U.S. Pat. No. 5,495,030; U.S. Pat. No.5,495,018; U.S. Pat. No. 5,494,903; U.S. Pat. No. 5,466,684; EP 0535346B1; U.S. Pat. No. 5,457,096; U.S. Pat. No. 5,442,059; U.S. Pat. No.5,430,162; U.S. Pat. No. 5,420,272; U.S. Pat. No. 5,401,863; U.S. Pat.No. 5,401,729; U.S. Pat. No. 5,386,041; U.S. Pat. No. 5,380,888; U.S.Pat. No. 5,371,076; EP 618 190; U.S. Pat. No. 5,326,759; EP 582 829; EP528 810; EP 582 790; EP 582 789; EP 582 788; U.S. Pat. No. 5,281,628; EP536 515; EP 535 346; WO 96/34852; WO 95/22529A1; U.S. Pat. No.4,066,694; U.S. Pat. No. 3,862,225; U.S. Pat. No. 3,622,627; WO01/87823A1; and WO 00/28983A1. Each of these aforementioned applicationsand patents are hereby incorporated herein by reference in its entirety.In addition, the invention pertains to each of the compounds describedherein, methods of using each of the compounds, and pharmaceuticalcompositions comprising each of the compounds.

The term “substituted tetracycline compound” includes tetracyclinecompounds with one or more additional substituents, e.g., at the 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a or 13 position or at any otherposition which allows the substituted tetracycline compound of theinvention to perform its intended function, e.g., to modulate RNA ortreat a DTMR. In certain embodiments, the substituted tetracyclinecompound is a 7-substituted sancycline compound, a 9-substitutedminocycline compound, or a 7,9-substituted sancycline compound. Incertain embodiments, the term “substituted tetracycline compound” doesnot include tetracycline compounds with a chlorine, hydrogen ordimethylamino substituent at the 7-position. In other embodiments, theterm “substituted tetracycline compound” does not include compounds witha hydrogen as a 9-position substituent. In other embodiments, the termsubstituted tetracycline does not include 5-hydroxy tetracycline,7-chlorotetracycline, 6-demethyl-7-chlorotetracycline,anhydrochlorotetracycline, 4-epi-anhydrochlorotetracycline, orβ-chelocardin.

The term “substituted tetracycline compound” also includes substitutedtetracycline compounds of the formula (I):

wherein

-   -   R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen,        alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen, alkyl, alkenyl,        alkynyl, aryl, substituted carbonyl, or a pro-drug moiety;    -   R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen,        or hydrogen;    -   R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl,        aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,        alkyl carbonyloxy, or aryl carbonyloxy;    -   R⁶ and R^(6′) are each independently hydrogen, methylene,        absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or        an arylalkyl;    -   R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,        alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,        arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,        heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a);    -   R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,        alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,        alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,        heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(8c)C(=E′)ER^(8a);    -   R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,        alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,        arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,        heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);    -   R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b),        R^(8c), R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d),        R^(9e), and R^(8f) are each independently hydrogen, acyl, alkyl,        alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   E is CR^(8d)R^(8e), S, NR^(8b) or O;    -   E′ is O, NR^(8f), or S;    -   W is CR^(7d)R^(7e), S, NR^(7b) or O;    -   W′ is O, NR^(7f), or S;    -   X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;    -   Y′ and Y are each independently hydrogen, halogen, hydroxyl,        cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl;    -   Z is CR^(9d)R^(9e), S, NR^(9b) or O;    -   Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts,        esters and enantiomers thereof.

In a further embodiment, the substituted tetracycline compounds offormula (I) comprise compounds wherein R², R^(2′), R⁸, R¹⁰, R¹¹, and R¹²are each hydrogen, X is CR⁶R^(6′), and R⁴ is NR^(4′)R^(4″), whereinR^(4′) and R^(4″) are each methyl. In addition, R⁹ may be hydrogen.

In one embodiment, R⁷ is substituted or unsubstituted aryl, e.g., phenylor heteroaryl. In a further embodiment, R⁷ is substituted with one ormore substituents which allow the substituted tetracycline compound toperform its intended function, e.g., treat a DTMR or modulate RNA.Examples of such substituents include alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino, acylamino, amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, aryl or heterocyclic moiety.

In another embodiment, R⁷ is substituted or unsubstituted alkenyl.Examples of substituents for alkenyl R⁷ groups include alkyl, alkenyl,alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, aryl orheterocyclic moiety.

In another embodiment, R⁷ is substituted or unsubstituted heteroaryl andR⁹ is alkyl.

In another further embodiment, the substituted tetracycline compound isa substituted minocycline compound, e.g., R⁷ is dialkylamino. In afurther embodiment, R⁹ is alkylamino. In another embodiment, R⁹ is—NR^(9c)C(═Z′)ZR^(9a), wherein R^(9c) is hydrogen, Z′ is nitrogen oroxygen, Z is NH, and R^(9a) is aryl or aralkyl.

Examples of tetracycline compounds include:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.Other examples of substituted tetracycline compounds are shown in Table2, below.

TABLE 2

In certain embodiments, the substituted tetracycline compounds of theinvention have antibacterial activity against gram + and/or gram −bacteria. In certain embodiments, the tetracycline compounds of theinvention do not have antibacterial activity against gram + and/or gram− bacteria. In other embodiments, compounds with MIC of greater thanabout 2 μg/ml, greater than about 3 μg/ml, greater than about 4 μg/ml,greater than about 5 μg/ml, greater than about 6 μg/ml, greater thanabout 8 μg/ml, greater than about 9 μg/ml, greater than about 10 μg/ml,greater than about 11 μg/ml, greater than about 12 μg/ml, greater thanabout 13 μg/ml, greater than about 14 μg/ml, greater than about 15μg/ml, greater than about 16 μg/ml, greater than about 17 μg/ml, greaterthan about 18 μg/ml, greater than about 19 μg/ml, greater than about 20μg/ml, greater than about 25 μg/ml, greater than about 30 μg/ml, greaterthan about 40 μg/ml, or greater than about 50 μg/ml for gram + and/orgram − bacteria are considered not to have anti-bacterial activity.

In other embodiments, compounds with MIC of less than about 50 μg/ml,less than about 40 μg/ml, less than about 30 μg/ml, less than about 25μg/ml, less than about 20 μg/ml, less than about 15 μg/ml, less thanabout 14 μg/ml, less than about 13 μg/ml, less than about 12 μg/ml, lessthan about 11 μg/ml, less than about 10 μg/ml, less than about 9 μg/ml,less than about 8 μg/ml, less than about 6 μg/ml, less than about 5μg/ml, less than about 4 μg/ml, less than about 3 μg/ml, less than about2 μg/ml, less than about 1 μg/ml, or less than about 0.5 μg/ml forgram + and/or gram − bacteria are considered to have anti-bacterialactivity.

In one embodiment, the tetracycline compound of the invention may retainantibiotic, antibacterial, or antimicrobial activity, it may havedecreased antibiotic, antibacterial, or antimicrobial activity, or, itmay have little to no antibiotic, antibacterial or antimicrobialactivity. In an embodiment, the substituted tetracycline compound issubstituted at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12aand/or 13 position. In certain embodiments, the tetracycline compoundsof the invention are 7 and/or 9 substituted, e.g., 7 and/or9-substituted tetracycline compounds (e.g., compounds wherein R⁷ and/orR⁹ are not both hydrogen). In yet a further embodiment, the tetracyclinecompounds of the invention are 7 and/or 9 substituted sancyclinecompounds. Other examples of tetracycline compounds which may be used inthe methods of the invention include those otherwise described herein orincorporated by reference.

The substituted tetracycline compounds of the invention can besynthesized using the methods described in Example 1, in the followingschemes and/or by using art recognized techniques. All novel substitutedtetracycline compounds described herein are included in the invention ascompounds.

9- and 7-substituted tetracyclines can be synthesized by the methodshown in Scheme 1. As shown in Scheme 1,9- and 7-substitutedtetracycline compounds can be synthesized by treating a tetracyclinecompound (e.g., doxycycline, 1A), with sulfuric acid and sodium nitrate.The resulting product is a mixture of the 7-nitro and 9-nitro isomers(1B and 1C, respectively). The 7-nitro (1B) and 9-nitro (1C) derivativesare treated by hydrogenation using hydrogen gas and a platinum catalystto yield amines 1D and 1E. The isomers are separated at this time byconventional methods. To synthesize 7- or 9-substituted alkenylderivatives, the 7- or 9-amino tetracycline compound (1E and 1F,respectively) is treated with HONO, to yield the diazonium salt (1G and1H). The salt (1G and 1H) is treated with an appropriate reactivereagent to yield the desired compound (e.g., in Scheme1,7-cyclopent-1-enyl doxycycline (1H) and 9-cyclopent-1-enyl doxycycline(1I)).

As shown in Scheme 2, tetracycline compounds of the invention wherein R⁷is a carbamate or a urea derivative can be synthesized using thefollowing protocol. Sancycline (2A) is treated with NaNO₂ under acidicconditions forming 7-nitro sancycline (2B) in a mixture of positionalisomers. 7-nitrosancycline (2B) is then treated with H₂ gas and aplatinum catalyst to form the 7-amino sancycline derivative (2C). Toform the urea derivative (2E), isocyanate (2D) is reacted with the7-amino sancycline derivative (2C). To form the carbamate (2G), theappropriate acid chloride ester (2F) is reacted with 2C.

As shown in Scheme 3, tetracycline compounds of the invention, whereinR⁷ is a heterocyclic (i.e. thiazole) substituted amino group can besynthesized using the above protocol. 7-amino sancycline (3A) is reactedwith Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).The protected thiourea (3C) is then deprotected yielding the activesancycline thiourea (3D) compound. The sancycline thiourea (3D) isreacted with an α-haloketone (3E) to produce a thiazole substituted7-amino sancycline (3F).

7-alkenyl tetracycline compounds, such as 7-alkynyl sancycline (4A) and7-alkenyl sancycline (4B), can be hydrogenated to form 7-alkylsubstituted tetracycline compounds (e.g., 7-alkyl sancycline, 4C).Scheme 4 depicts the selective hydrogenation of the 7-position double ortriple bond, in saturated methanol and hydrochloric acid solution with apalladium/carbon catalyst under pressure, to yield the product.

In Scheme 5, a general synthetic scheme for synthesizing 7-position arylderivatives is shown. A Suzuki coupling of an aryl boronic acid with aniodosancycline compound is shown. An iodo sancycline compound (5B) canbe synthesized from sancycline by treating sancycline (5A) with at leastone equivalent N-iodosuccinimide (NIS) under acidic conditions. Thereaction is quenched, and the resulting 7-iodo sancycline (5B) can thenbe purified using standard techniques known in the art. To form the arylderivative, 7-iodo sancycline (5B) is treated with an aqueous base(e.g., Na₂CO₃) and an appropriate boronic acid (5C) and under an inertatmosphere. The reaction is catalyzed with a palladium catalyst (e.g.,Pd(OAc)₂). The product (5D) can be purified by methods known in the art(such as HPLC). Other 7-aryl, alkenyl, and alkynyl tetracyclinecompounds can be synthesized using similar protocols.

The 7-substituted tetracycline compounds of the invention can also besynthesized using Stille cross couplings. Stille cross couplings can beperformed using an appropriate tin reagent (e.g., R—SnBu₃) and ahalogenated tetracycline compound, (e.g., 7-iodosancycline). The tinreagent and the iodosancycline compound can be treated with a palladiumcatalyst (e.g., Pd(PPh₃)₂Cl₂ or Pd(AsPh₃)₂Cl₂) and, optionally, with anadditional copper salt, e.g., CuI. The resulting compound can then bepurified using techniques known in the art.

The compounds of the invention can also be synthesized using Heck-typecross coupling reactions. As shown in Scheme 6, Heck-typecross-couplings can be performed by suspending a halogenatedtetracycline compound (e.g., 7-iodosancycline, 6A) and an appropriatepalladium or other transition metal catalyst (e.g., Pd(OAc)₂ and CuI) inan appropriate solvent (e.g., degassed acetonitrile). The substrate, areactive alkene (6B) or alkyne (6D), and triethylamine are then addedand the mixture is heated for several hours, before being cooled to roomtemperature. The resulting 7-substituted alkenyl (6C) or 7-substitutedalkynyl (6E) tetracycline compound can then be purified using techniquesknown in the art.

To prepare 7-(2′-Chloro-alkenyl)-tetracycline compounds, the appropriate7-(alkynyl)-sancycline (7A) is dissolved in saturated methanol andhydrochloric acid and stirred. The solvent is then removed to yield theproduct (7B).

As depicted in Scheme 8,5-esters of 9-substituted tetracycline compoundscan be formed by dissolving the 9-substituted compounds (8A) in strongacid (e.g. HF, methanesulphonic acid, and trifluoromethanesulfonic acid)and adding the appropriate carboxylic acid to yield the correspondingesters (8B).

As shown in Scheme 9, methacycline (9A) can be reacted with aphenylboronic acid in the presence of a palladium catalyst such asPd(OAc)₂ to form a 13 aryl substituted methacycline compound. Theresulting compound can then be purified using techniques known in theart such as preparative HPLC and characterized.

As shown in Scheme 10 below, 7 and 9 aminomethyl tetracyclines may besynthesized using reagents such as hydroxymethyl-carbamic acid benzylester. The resulting aminomethyl tetracycline compounds may be furtherderivatized

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic)groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone (e.g., C₁-C₂₀ for straight chain, C₃-C₂₀for branched chain), and more preferably 4 or fewer. Cycloalkyls mayhave from 3-8 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure. The term C₁-C₆ includes alkylgroups containing 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxophenyl, quinoline, isoquinoline, naphthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 20 or fewer carbon atoms inits backbone (e.g., C₂-C₂₀ for straight chain, C₃-C₂₀ for branchedchain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms intheir ring structure, and more preferably have 5 or 6 carbons in thering structure. The term C₂-C₂₀ includes alkenyl groups containing 2 to20 carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 20 or fewer carbon atoms in its backbone (e.g., C₂-C₂₀for straight chain, C₃-C₂₀ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including, e.g., alkylcarbonylamino, arylcarbonylamino,carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkenyl, alkynyl groups,halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “amide” or “aminocarboxy” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻X⁺,where X⁺ is a counterion.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “oximyl” includes moieties which comprise an oxime group.

The term “dimeric moiety” includes moieties which comprise a secondtetracycline four ring structure. The dimeric moiety may be attached tothe substituted tetracycline through a chain of from 1-30 atoms. Thechain may be comprised of atoms covalently linked together throughsingle, double and triple bonds. The tetracycline ring structure of thedimeric moiety may further be substituted or unsubstituted. It may beattached at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a, and/or13 position.

The term “prodrug moiety” includes moieties which can be metabolized invivo. Generally, the prodrugs moieties are metabolized in vivo byesterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters. Prodrugs which are converted to activeforms through other mechanisms in vivo are also included.

The structures of some of the substituted tetracycline compounds used inthe methods and compositions of the invention include asymmetric carbonatoms. The isomers arising from the chiral atoms (e.g., all enantiomersand diastereomers) are included within the scope of this invention,unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof.

The method may further comprise administering the tetracycline compoundin combination with a second agent, e.g., an agent which may enhancetreatment of the DTMR, enhance the modulation of RNA, or the secondagent may be selected for treating a different DTMR or a differentdisease state not related to the RNA modulation.

The language “in combination with” a second agent includesco-administration of the tetracycline compound, and with the secondagent, administration of the tetracycline compound first, followed bythe second agent and administration of the second agent first, followedby the tetracycline compound. The second agent may be any agent which isknown in the art to treat, prevent, or reduce the symptoms of a DTMR.Furthermore, the second agent may be any agent of benefit to the patientwhen administered in combination with the administration of antetracycline compound. Examples of second agents include neuroprotectiveagents and chemotherapeutic agents.

The language “chemotherapeutic agent” includes chemical reagents whichinhibit the growth of proliferating cells or tissues wherein the growthof such cells or tissues is undesirable or otherwise treat at least oneresulting symptom of such a growth. Chemotherapeutic agents are wellknown in the art (see e.g., Gilman A. G., et al., The PharmacologicalBasis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and aretypically used to treat neoplastic diseases. Examples ofchemotherapeutic agents include: bleomycin, docetaxel (Taxotere),doxorubicin, edatrexate, etoposide, finasteride (Proscar), flutamide(Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex),granisetron (Kytril), irinotecan (Campto/Camptosar), ondansetron(Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpinehydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2(Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab(Herceptin), tretinoin (Retin-A), Triapine, vincristine, and vinorelbinetartrate (Navelbine).

Other examples of chemotherapeutic agents include alkylating drugs suchas Nitrogen Mustards (e.g., Mechlorethamine (HN₂), Cyclophosphamide,Ifosfamide, Melphalan (L-sarcolysin), Chlorambucil, etc.);ethylenimines, methylmelamines (e.g., Hexamethylmelamine, Thiotepa,etc.); Alkyl Sulfonates (e.g., Busulfan, etc.), Nitrosoureas (e.g.,Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU),Streptozocin (streptozotocin), etc.), triazenes (e.g., Decarbazine(DTIC; dimethyltriazenoimi-dazolecarboxamide)), Alkylators (e.g.,cis-diamminedichloroplatinum II (CDDP)), etc.

Other examples of chemotherapeutic agents include antimetabolites suchas folic acid analogs (e.g., Methotrexate (amethopterin)); pyrimidineanalogs (e.g., fluorouracil (′5-fluorouracil; 5-FU); floxuridine(fluorode-oxyuridine); FUdr; Cytarabine (cyosine arabinoside), etc.);purine analogs (e.g., Mercaptopurine (6-mercaptopurine; 6-MP);Thioguanine (6-thioguanine; TG); and Pentostatin (2′-deoxycoformycin)),etc.

Other examples of chemotherapeutic agents also include vinca alkaloids(e.g., Vinblastin (VLB) and Vincristine); topoisomerase inhibitors(e.g., Etoposide, Teniposide, Camptothecin, Topotecan,9-amino-campotothecin CPT-11, etc.); antibiotics (e.g., Dactinomycin(actinomycin D), adriamycin, daunorubicin, doxorubicin, bleomycin,plicamycin (mithramycin), mitomycin (mitomycin C), Taxol, Taxotere,etc.); enzymes (e.g., L-Asparaginase); and biological response modifiers(e.g., interferon-; interleukin 2, etc.). Other chemotherapeutic agentsinclude cis-diaminedichloroplatinum II (CDDP); Carboplatin;Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine(N-methylhydrazine); and adrenocortical suppressants (e.g., Mitotane,aminoglutethimide, etc.).

Other chemotherapeutic agents include adrenocorticosteroids (e.g.,Prednisone); progestins (e.g., Hydroxyprogesterone caproate;Medroxyprogesterone acetate, Megestrol acetate, etc.); estrogens (e.g.,diethylstilbestrol; ethenyl estradiol, etc.); antiestrogens (e.g.Tamoxifen, etc.); androgens (e.g., testosterone propionate,Fluoxymesterone, etc.); antiandrogens (e.g., Flutamide); andgonadotropin-releasing hormone analogs (e.g., Leuprolide).

III. Pharmaceutical Compositions for the Treatment of DTMR

The invention also pertains at least in part to pharmaceuticalcompositions for the treatment of DTMR. The pharmaceutical compositionscomprise a tetracycline compound of the invention in combination with apharmaceutical acceptable carrier. The composition may further comprisea second agent for the treatment of a DTMR.

The language “pharmaceutical composition” includes preparations suitablefor administration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal,pulmonary and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane. Sprays also can be delivered by mechanical,electrical, or by other methods known in the art.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial, antiparasitic and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle. The compositions also may be formulated such that itselimination is retarded by methods known in the art.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration or administration via inhalation ispreferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually. Other methods foradministration include via inhalation.

The tetracycline compounds of the invention may also be administered toa subject via stents. The compounds may be administered through thestent or be impregnated in the stent itself.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient willrange from about 0.0001 to about 100 mg per kilogram of body weight perday, more preferably from about 0.01 to about 50 mg per kg per day, andstill more preferably from about 1.0 to about 100 mg per kg per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition. Compounds or pharmaceutical compositions canbe administered in combination with other agents.

As set out above, certain embodiments of the present compounds cancontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” is art recognized and includes relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Farm. SCI.66:1-19).

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesincludes relatively non-toxic, inorganic and organic base addition saltsof compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. Hydroxyls canbe converted into esters via treatment with an esterifying agent such asalkanoyl halides. The term also includes lower hydrocarbon groupscapable of being solvated under physiological conditions, e.g., alkylesters, methyl, ethyl and propyl esters. (See, for example, Berge etal., supra.)

The invention also pertains, at least in part, to packaged compositionscomprising a tetracycline compound of the invention and instructions forusing said compound for the treatment of a DTMR.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXEMPLIFICATION OF THE INVENTION

Compounds of the invention may be made as described below, withmodifications to the procedure below within the skill of those ofordinary skill in the art.

Example 1 Synthesis of 7-Substituted Tetracyclines 7 Iodo Sancycline

One gram of sancycline was dissolved in 25 mL of TFA (trifluoroaceticacid) that was cooled to 0 C (on ice). 1.2 equivalents ofN-iodosuccinimide (NIS) was added to the reaction mixture and reactedfor forty minutes. The reaction was removed from the ice bath and wasallowed to react at room temperature for an additional five hours. Themixture was then analyzed by HPLC and TLC, was driven to completion bythe stepwise addition of NIS. After completion of the reaction, the TFAwas removed in vacuo and 3 mL of MeOH was added to dissolve the residue.The methanolic solution was the added slowly to a rapidly stirringsolution of diethyl ether to form a greenish brown precipitate. The7-iodo isomer of sancycline was purified by treating the 7-iodo productwith activated charcoal, filtering through Celite, and subsequentremoval of the solvent in vacuo to produce the 7-isomer compound as apure yellow solid in 75% yield.

MS (M+H) (formic acid solvent) 541.3.

\Rt: Hypersil C18 BDS Column, 11.73

¹H NMR (Methanol d₄-300 MHz) δ 7.87-7.90 (d, 1H), 6.66-6.69 (d, 1H),4.06 (s, 1H), 2.98 (s, 6H), 2.42 (m, 1H), 2.19 (m, 1H), 1.62 (m, 4H),0.99 (m, 2H).

7-Phenyl Sancycline

7-iodosancycline, 150 mg (0.28 mM), Pd(OAc)₂ and 10 mL of MeOH are addedto a flask with a stir bar and the system degassed 3× using argon.Na₂CO₃ (87 mg, 0.8 mM) dissolved in water and argon degassed is addedvia syringe is added along with phenylboronic acid (68 mg, 0.55 mM) inMeOH that was also degassed. The reaction was followed by HPLC for 2hours and cooled to room temperature. The solution was filtered, anddried to produce a crude mixture. The solid was dissolved indimethylformamide and injected onto a preparative HPLC system using C18reverse-phase silica. The fraction at 36-38 minutes was isolated, andthe solvent removed in vacuo to yield the product plus salts. The saltswere removed by extraction into 50:25:25 water, butanol, ethyl acetateand dried in vacuo. This solid was dissolved in MeOH and the HCl saltmade by bubbling in HCl gas. The solvent was removed to produce theproduct in 42% yield as a yellow solid.

Rt 21.6 min: MS (M+H, formic acid solvent): 491.3

¹H NMR (Methanol d₄-300 MHz) δ 7.87 (d, J=8.86 Hz, 1H), 7.38 (m, 5H),6.64 (d, 8.87 Hz, 1H), 4.00 (s, 1H), 3.84 (s, 2H), 3.01 (s, 6H), 2.46(m, 2H), 1.63 (m, 4H), 0.95 (m, 2H).

7-(4′-Chlorophenyl)Sancycline

7-iodosancycline, 500 mg (0.91 mM), Pd(OAc)₂ 21 mg, and 20 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Na₂CO₃ (293 mg, 2.8 mM) dissolved in water and argon degassed isadded via syringe is added along with 4-C1-phenylboronic acid (289 mg,1.85 mM) in MeOH that was also degassed. The reaction was followed byHPLC for 45 minutes and cooled to room temperature. The solution wasfiltered, and dried to produce a crude mixture. The solid was dissolvedin dimethylformamide and injected onto a preparative HPLC system usingC18 reverse-phase silica. The fraction at 39 minutes was isolated, andthe solvent removed in vacuo to yield the product plus salts. The saltswere removed by extraction into 50:25:25 water, butanol, ethyl acetateand dried in vacuo. This solid was dissolved in MeOH and the HCl saltmade by bubbling in HCl gas. The solvent was removed to produce theproduct in 57% yield as a yellow solid.

Rt 20.3 min: MS (M+H, formic acid solvent): 525.7

¹H NMR (Methanol d₄-300 MHz) δ 7.49-7.52 (d, J=8.54 Hz, 1H), 6.99-7.01(d, 8.61 Hz, 1H), 4.12 (s, 1H), 3.67 (m, 1H), 3.06 (s, 6H), 2.58 (m,2H), 1.62 (m, 4H), 1.01 (m, 2H).

7-(4′-Fluorophenyl)Sancycline

7-iodosancycline, 200 mg (0.3 mM), Pd(OAc)₂ 8.3 mg, and 10 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Na₂CO₃ (104 mg, 1.1 mM) dissolved in water and argon degassed isadded via syringe is added along with 4-F-phenylboronic acid (104 mg,0.7 mM) in MeOH that was also degassed. The reaction was followed byHPLC for 20 minutes and cooled to room temperature. The solution wasfiltered, and dried to produce a crude mixture. The solid was dissolvedin dimethylformamide and injected onto a preparative HPLC system usingC18 reverse-phase silica. The fraction at 19-20 minutes was isolated,and the solvent removed in vacuo to yield the product plus salts. Thesalts were removed by extraction into 50:25:25 water, butanol, ethylacetate and dried in vacuo. This solid was dissolved in MeOH and the HClsalt made by bubbling in HCl gas. The solvent was removed to produce theproduct in 47% yield as a yellow solid.

Rt 19.5 min: MS (M+H, formic acid solvent): 509.4

¹H NMR (Methanol d₄-300 MHz) δ 6.92-6.95 (d, 1H), 7.45-7.48 (d, 1H),7.15-7.35 (m, 4H), 4.05 (s, 1H), 3.62 (m, 1H), 3.08 (s, 6H), 2.55 (m,2H), 1.65 (m, 4H), 1.00 (m, 2H).

7-(4′-Iodo-1′,3′-carboethoxy-1′,3′-butadiene)Sancycline

7-I-Sancycline (1 gm, 1.86 mmol), was dissolved in 25 mL of acetonitrileand was degassed and purged with nitrogen (three times). To thissuspension Pd(OAc)_(Z) (20 mg, 0.089 mmol), CuI (10 mg, 0.053 mmol),(o-tolyl)₃P (56 mg, 0.183 mmol) were added and purged with nitrogen.Ethyl propiolate (1 mL) and triethylamine (1 mL) were added to thesuspension. It turned to a brown solution upon addition of Et₃N. Thereaction mixture was then heated to 70 degrees C. for two hours.Progress of the reaction was monitored by HPLC. It was then cooled downto room temperature and was filtered through celite. Evaporation of thesolvent gave a brown solid, which was then purified on preparative HPLCto give a yellow solid.

7-(2′-Chloroethenyl)-Sancycline

To a solution/suspension of 0.65 g (1 mmol) of 7-iodo sancycline, 0.05 gtetrakis triphenyl phosphinato palladate, 0.012 g palladium acetate,0.05 g copper (I) iodide in 10 mL acetonitrile, 2 mL triethylamine and0.5 g trimethylsilyl acetylene was added at room temperature. Thereaction proceeded for two hours before being filtered through a celitebed and concentrated. The crude product was purified by preparativeHPLC. The collected fractions were concentrated and the residue wastaken up in about 1 mL of methanol and 2 mL of HCl saturated methanol.The product was precipitated with ether. The solids were filtered offand dried under reduced pressure. NMR spectroscopy and LC-MS showed thatthe compound was 7-(2-chloroethenyl) sancycline.

7-(4′-aminophenyl)Sancycline

To a solution of 200 mg of 7-(4-nitrophenyl)sancycline in 50 mLmethanol, 10 mg of 10% palladium on charcoal catalyst was added. Thereaction mixture was shaken under 40 psi hydrogen pressure for 2 hoursand was then filtered followed by concentration. The residue was furtherpurified by preparative HPLC. 35 mg was isolated as the HCl salt and thestructure was proved by NMR and LC-MS to be 7-(4-aminophenyl)sancycline.

7-(N,N-Dimethylpropynyl)-Sancycline

7-I-Sancycline (1 gm, 1.86 mmol), taken in 25 mL of acetonitrile wasdegassed and purged with nitrogen (three times). To this suspensionPd(OAc)₂ (20 mg, 0.089 mmol), CuI (10 mg, 0.053 mmol), (o-tolyl)₃P (56mg, 0.183 mmol) were added and purged with nitrogen for few minutes.N,N-Dimethylpropyne (308 mg, 3.72 mmol) and triethylamine (1 mL) wereadded to the suspension. It was turned into a brown solution uponaddition of Et₃N. The reaction mixture was then heated to 70° C. for 3hours. Progress of the reaction was monitored by HPLC. It was thencooled down to room temperature and was filtered through celite.Evaporation of the solvent gave a brown solid, which was then purifiedon preparative HPLC to give a yellow solid. The structure of thiscompound has been characterized using 1H NMR, HPLC, and MS.

7-(2′-Chloro-3-Hydroxypropenyl)-Sancycline

7-(alkynyl)-sancycline (100 mg) was taken in 20 ml of saturated MeOH/HCland stirred for 20 min. The solvent was then evaporated to give a yellowpowder. The structure of this compound has been characterized using ¹HNMR, HPLC, and MS.

7-(3′-Methoxyphenylethyl)-Sancycline

7-(3′-Methoxyphenylethynyl)-sancycline (1 mmol) was taken in saturatedsolution of MeOH/HCl. To this solution 10% Pd/C was added and wassubjected to hydrogenation at 50 psi for 12 hrs. It was then filteredthrough celite. The solvent was evaporated to give a yellow powder.Finally, it was precipitated from MeOH/diethylether. The structure ofthis compound has been characterized using 1H NMR, HPLC, and MS.

(2-Dimethylamino-Acetylamino)-Sancycline

N,N-Dimethylglycine (1.2 mmol) was dissolved in DMF (5 mL) andO-Benzotriazol-1-yl-N,N,N′,N′,-tetramethyluronium hexafluorophosphate(HBTU, 1.2 mmol) was added. The solution was then stirred for 5 minutesat room temperature. To this solution, 7-aminosancycline (1 mmol) wasadded, followed by the addition of diisopropylethyl amine (DIEA, 1.2mmol). The reaction was then stirred at room temperature for 2 hours.The solvent, DMF, was removed on vacuum. The crude material wasdissolved in 5 mL of MeOH and filtered using autovials and purifiedusing preparative HPLC. The structure of the product has beencharacterized using 1H NMR, HPLC, and MS.

7-(N-Methylsulphonamidopropargylamine)Sancycline

To a mixture of 7-iodosancycline mono trifluoroacetic acid salt (1 g;1.53 mmoles), palladium II acetate (17.2 mg; 0.076 mmoles), tetrakistriphenylphosphine palladium (176.8 mg; 0.153 mmoles), and copper (I)iodide (49 mg; 0.228 mmoles) was added 15 ml of reagent gradeacetonitrile in a clean dry 2 necked round bottom flask. The reactionwas purged with a slow steam of argon gas, with stirring, for 5 minutesbefore the addition (in one portion as a solid) ofN-methylsulphonamidopropargyl amine. The sulphonamide was prepared by amethod known in the art (J. Med. Chem. 31(3) 1988; 577-82). This wasfollowed by one milliliter of triethylamine (1 ml; 0.726 mg; 7.175mmoles) and the reaction was stirred, under an argon atmosphere, forapproximately 1.0 hour at ambient temperature. The reaction mixture wassuctioned filtered through a pad of diatomaceous earth and washed withacetonitrile. The filtrates were reduced to dryness under vacuo and theresidue was treated with a dilute solution of trifluororoacetic acid inacetonitrile to adjust the pH to approximately 2. The residue wastreated with more dilute trifluoroacetic acid in acetonitrile, resultingin the formation of a precipitate, which was removed via suctionfiltration. The crude filtrates were purified utilizing reverse phaseHPLC with DVB as the solid phase; and a gradient of 1:1methanol/acetonitrile 1% trifluoroacetic acid and 1% trifluoroaceticacid in water. The appropriate fractions were reduced to dryness underreduced pressure and solid collected. The product was characterized via¹H NMR, mass spectrogram and LC reverse phase.

7-(2′-methoxy-5′-formylphenyl)sancycline

7-iodo-sancycline (1 g, 1.53 mmol), Pd(OAc)₂ (34 mg, 0.153 mmol), andMeOH (50 mL) were combined in a 250 mL 2 neck round bottom flaskequipped with a condenser and argon line. The solution was then purgedwith argon (15 min) while heated in an oil bath to approximately 70° C.Sodium carbonate (482 mg, 4.58 mmol) was dissolved in water (3-5 mL) andadded to reaction flask. The flask was then purged with argon foranother 5 minutes. 2-Methoxy-5-formylphenyl boronic acid (333 mg, 1.83mmol) was dissolved in MeOH (5 mL) and added to reaction flask. Theflask was then purged again with argon for 10 minutes. The reaction wasmonitored to completion within 3 hours. The contents of the flask werefiltered through filter paper and the remaining solvent was evacuated.To make the hydrochloric acid salt, the residue was dissolved in MeOH(sat. HCl) to make the HCl salt. The solution was then filtered and thesolvent was evacuated. The product was then characterized by ¹H NMR,LC-MS.

7-(2′-Methoxy-5′-N,N′-Dimethylaminomethylphenyl)Sancycline

7-(2′-methoxy-5′-formylphenyl)sancycline (1 g, 1.82 mmol), dimethylamineHCl (297 mg, 3.64 mmol), triethylamine (506 μL, 3.64 mmol), and 1,2-DCE(7 mL) were combined in a 40 mL vial. The contents were dissolved withinseveral minutes of shaking or stirring. Sodium triacetoxyborohydride(772 mg, 3.64 mmol) was then added as a solid. The reaction wasmonitored by HPLC and LC-MS and was complete within 3 hours. Thereaction was quenched with MeOH (20 mL) and the solvent was subsequentlyevacuated. The residue was redissolved in 3 mL DMF and separated on aC-18 column. Fractions from the prep column dried down in-vacuo and theHCl salt was made by dissolving contents in methanol (sat. HCl). Thesolvent was reduced and a yellow powder obtained. Characterized by ¹HNMR, LC-MS, HPLC.

7-Furanyl Sancycline

7-iodo sancycline (1.3 mg) and Pd(OAc)₂ were taken in 100 mL of methanoland purged with argon for five minutes at 70° C. To this solution wasadded a solution of sodium carbonate (44 mg) in water (previously purgedwith argon). A yellow precipitate was obtained and the mixture washeated for another ten minutes. 3-Furanyl boronic acid (333 mg, solutionin DMF, purged with argon) was then added and the mixture was heated foranother two hours at 70° C. The reaction was monitored by MPLC/MS. Whenthe reaction was complete, the mixture was filtered through celite andthe solvent was removed to give a crude material. The crude material waspurified by precipitating it with ether (200 ml). The yellow precipitatewas filtered and purified using preparative HPLC. The hydrochloride saltwas made by dissolving the material in MeOH/HCl and evaporating todryness. The identity of the resulting solid was confirmed using HPLC,MS, and NMR.

4S-(4α,12aα)]-4-Dimethylamino-7-ethynyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxamide

300 mg of 7-iodosancycline 6A was dissolved in 20 mL of acetonitrile and2.0 mL triethylamine, 50.0 mg Pd(PPh₃)₄, 50 mg CuI, 12.5 mg Pd(OAc)₂ wasadded followed by 0.5 mL of trimethylsilylacetylene. The reaction wasstirred at room temperature for 4 hours, filtered through adivinyl-benzene cartridge (25 g), and concentrated in vacuo to yield 280mg of the crude material (monitored by LC/MS). The TMS group was removedby dissolving the crude material in methanol, and adding 250 mg of K₂CO₃while stirring for 4 hours at room temperature to yield compound 6E(Scheme 11). The mixture was filtered through a divinylbenzenecartridge. The solvent was removed in vacuo to yield the 7-ethynylsancycline 6E (Scheme 11) in 60% yield by HPLC.

General Method for Synthesis of 7-acetyl sancycline and 7-carbonylalkylDerivatives of Sancycline

7-ethynyl sancycline 6E (Scheme 11, 300 mg) or ethynyl substitutedderivatives of 7-ethynyl sancycline are dissolved in 0.1 mL water, 2 mLof H₂SO₄, optionally with HgSO₄ (170 mg) and stirred overnight at roomtemperature. The aqueous layer is extracted into butanol, CH₂Cl₂ or anequivalent and the solvent is removed to yield the crude compound 11A(Scheme 11). 7-acetyl sancycline (11A, Scheme 11) is isolated via C18reverse-phase HPLC or by other methods in the art to yield pure compoundin good yield. M+H=457.4

Conversion of 7-acetyl or 7-carbonylalkyl Derivatives of Sancycline toOximes or O-alkyl Oximes

1 gram of 7-acetyl or 7-carbonylalkyl derivatives of sancycline 11A(Scheme 11, 2 mmol) and hydroxylamine HCl are dissolved in methanol orethanol and stirred at room temperature for 2 or more hours. Thecompounds are isolated as the syn and anti isomers appropriately bypreparative C18-HPLC or by other methods in the art to yield7-oximecarbonyl alkyl derivatives of sancycline or 7-O-substitutedoximecarbonyl derivatives in good yield. 7-acetyl-oxime (Scheme 11,11B); M+H=473.5. 11C 7-acetyl-oxime-O-methyl ether; M+H=487.5. The synor anti isomers are both attainable by fractionation of HPLC solventvolumes.

General Methods and Conversion of 7-Acetyl or 7-CarbonylalkylDerivatives of Sancycline to 7-Carbonyl-α-Amino Derivatives

1 gram of 7-acetyl or 7-carbonylalkyl derivatives of sancycline 11A(Scheme 11, 2 mmol) is reacted with bromine (4 mmol) or typicalhalogenating agent (NBS, NCS or equivalent, 2-4 mmol) to produce theα-halogenated derivative 11D (Br, Cl) as crude solid. This compound isisolated by extraction or other methods in the art and may be reactedwith nucleophilic amines (2-4 mmol) or other nucleophiles (C or O-based)to yield α-amino derivatives of 7-acetyl 11E or other 7-carbonylalkylderivatives of sancycline.

Example 2 Preparation of 9-Substituted Minocyclines Preparation of9-Iodominocycline

To 200 ml of 97% methanesulfonic acid was slowly added, at ambienttemperature, portionwise [30 g; 56.56 mM] ofminocycline-bis-hydrochloride salt. The dark yellow brown solution wasthen stirred at ambient temperature while [38 g; 169.7 mM] ofN-iodosuccinimide was added, in six equal portions, over 3.0 hours time.The reaction was monitored via analytical LC, noting the disappearanceof the starting material.

The reaction was slowly quenched into 2 L of ice cold water containing[17.88 g; 1134.1 mM] of sodium thiosulfate with rapid stirring. Thisquench was stirred for approximately 30 minutes at ambient temperature.The aqueous layer was then extracted with 6×200 ml of ethyl acetatebefore the aqueous was poured onto [259.8 g; 3.08M] of sodium hydrogencarbonate containing 300 ml of n-butanol. The phases were split and theaqueous extracted with 4×250 ml of n-butanol. The organic fractions werecombined and washed with 3×250 ml of water and once with 250 ml ofsaturated brine. The resulting organic phase was reduced to drynessunder reduced pressure. The residue was suspended in methanol (˜600 ml)and anhydrous HCl gas was bubbled into this mixture until solutionoccurred This solution was reduced to dryness under reduced pressure.The filtrates were reduced to dryness under reduced pressure. Theresulting material was triturated with 300 ml of methyl t-butyl etherand isolated via filtration. This material was redissolved in 300 ml ofmethanol and treated with 0.5 g of wood carbon, filtered and filtratesreduced to dryness under reduced pressure. The material was againpowdered under methyl t-butyl ether, isolated via suction filtration andwashed with more ether, and finally hexanes. The material was vacuumdried to give 22.6 g of a light yellow brown powder.

General Procedure for Preparation of 9-Alkynyl Minocycline Compounds

1 mmol 9-iodo minocycline, 50 mg tetrakis triphenylphosphinatopalladate, 12 mg palladium acetate, 32 mg copper (I) iodide aredissolved/suspended in 10 ml acetonitrile. 2 to 5 ml triethylamine and 3to 5 mmol alkynyl derivative is added. The reaction mixture isvigorously stirred between ambient temperature to 70° C. The reactiontime is 2-24 hours. When the reaction is completed the dark suspensionis filtered through a celite bed and concentrated. The crude product ispurified by prep HPLC. The combined fractions are concentrated and takenup in ˜1 ml methanol. ˜3 ml HCl saturated methanol is added, and theproduct is precipitated with ether.

General Procedure for Preparation of 9-Aryl Minocycline Compounds

0.15 mmol of 9-iodominocycline, PdOAc (3.2 mg), 229 μl 2M Na₂CO₃ and 2equivalents of phenyl boronic acid were dissolved/suspended in 10 mlmethanol. The reaction flask was purged with argon and the reaction runfor a minimum of four hours or until HPLC monitoring shows consumptionof starting material and/or the appearance of products. The suspensionwas filtered through celite, and subject to purification by prep HPLC ona divinylbenzene or CIE reverse-phase column.

9-(4-Trifluoromethoxyphenylureido)-Methyl Minocycline

To 3 mL of dimethylformamide was added 150 mg (0.25 mmol) of 9-methylaminominocyline trihydrochloride and 67 mL (0.50 mmol) of triethylamineat 25° C. With stirring, 75 mL (0.50 mmol) of4-trifluoromethoxyphenylisocyanate was added and the resulting reactionmixture was stirred at 25° C. for two hours. The reaction was monitoredby analytical HPLC (4.6×50 mm reversed phase Luna C18 column, 5 minutelinear gradient 1-100% B buffer, A buffer was water with 0.1%trifluoroacetic acid, B buffer was acetonitrile with 0.1%trifluoroacetic acid). Upon completion, the reaction was quenched with 1mL of water and the pH adjusted to approximately 2.0 with concentratedHCl. The solution was filtered and the compound purified by preparativeHPLC. The product yield was 64 mg (37% yield). The purity of the productwas 95%, as determined by LCMS (M+1=690).

9-(4′Carboxy phenyl) Minocycline

In a clean, dry reaction vessel, was placed 9-iodominocycline [500 mg;0.762 mmoles] bis HCl salt, palladium (II) acetate [17.2 mg; 0.076mmoles] along with 10 ml of reagent grade methanol. The solution wasimmediately purged, with stirring, with a stream of argon gas forapproximately 5 minutes. The reaction vessel was brought to reflux andto it was sequentially added via syringe 2M potassium carbonate solution[1.91 ml; 3.81 mmoles], followed by a solution of p-carboxyphenylboronic acid [238.3 mg; 1.53 mmoles] in 5 ml of reagent DMF. Both ofthese solutions were previously degassed with argon gas forapproximately 5 minutes. The reaction was heated for 45 minutes, theprogress was monitored via reverse phase HPLC. The reaction wassuctioned filtered through a pad of diatomaceous earth and washed withDMF. The filtrates were reduced to an oil under vacuum and residuetreated with t-butylmethyl ether. Crude material was purified viareverse phase HPLC on DVB utilizing a gradient of water andmethanol/acetonitrile containing 1.0% trifluoroacetic acid. Productconfirmed by mass spectrum: found M+1 578.58; the structure corroboratedwith 1H NMR.

Example 3 Modulation of Murine Macrophage mRNAs Using TetracyclinesMaterials and Methods

Two murine macrophage cell lines were used: J774.2 (gift from PeterLambert, Aston University, UK), and RAW 264.7 (ATCC item number TIB-71).Cells were harvested from nearly confluent culture flasks and seededinto 6 well plates at a density of 5×10⁶ cells well⁻¹ in a volume of 3ml Dulbecco's modified essential medium supplemented with 10% fetal calfserum. After 2 hours, cells were exposed to the following conditions:

1) control (J774 cells on two separate occasions, and RAW 264.7 cells)

2) 50 μg/ml minocycline (J774 cells on two separate occasions, and RAW264.7 cells)

3) 100 ng/ml LPS (J774 cells on two separate occasions, and RAW 264.7cells)

4) 50 μg/ml minocycline+100 ng/ml LPS (J774 cells on two separateoccasions, and RAW 264.7 cells)

5) 50 μg/ml Compound A+100 ng/ml LPS (J774 cells only)

6) 50 μg/ml Compound B+100 ng/ml LPS (J774 cells only)

The tetracycline compounds were added 1.5 hours post seeding, thirtyminutes before the addition of LPS. The plates were incubated at 37° C.at 5% CO₂ in a humidified incubator.

Sample Processing, Hybridization, and Scanning

24 hours after incubation under experimental conditions, media wasremoved from the wells, and total RNA was purified from each sampleusing QIAGEN RNeasy® Mini columns. The manipulations which were thenperformed on the total RNA samples were as outlined in The Affymetrix®GeneChip® Expression Analysis technical manual, sections 2, chapter 1,entitled Eukaryotic Target Preparation. Briefly, RNA was reversetranscribed into double stranded cDNA with an oligo dT primer containinga T7 promoter. The product was then purified byphenol:chloroform:isoamyl extraction and ethanol precipitation, and thenused in an in vitro translation reaction to synthesize biotin-labelledantisense cRNA (Affymetrix controls of B. subtilis genes excised frompBluescript plasmid with Xho I digestion were added at this stage tocontrol for correct translation and biotin incorporation). The cRNA wasthen cleaned using QIAGEN RNeasy® Mini columns, and the resulting cRNAsolution fragmented using metal-induced hydrolysis.

Samples were prepared for hybridization with the Affymetrix murinegenome chips U74AV2 according to the directions in the Affymetrix®GeneChip® Expression Analysis technical manual, sections 2, chapters 3and 4, entitled Eukaryotic Target Hybridization and Eukaryotic Arrays:Washing, Staining and Scanning. Briefly, 15 ug Fragmented cRNA was mixedwith Affymetrix hybridization controls, herring sperm DNA, BSA, andconcentrated hybridization buffer, boiled for 5 minutes, centrifuged at14000×g for 5 minutes to obtain a precipitated-free solution, andhybridized with the array for 16 hours. Following hybridization, theAffymetrix washing and staining procedure was used entitled Washing andStaining Procedure 2: Antibody Amplification for Eukaryotic Targets.

Data Analysis

I. Finding mRNAs which are Up-Regulated or Down-Regulated by Minocycline

For both J774.2 and RAW264.7 cells, two lists were generated, one ofmRNAs which were increased at lease 2-fold by minocycline, and one ofmRNAs which were decreased at least 2-fold by minocycline. In the caseof mRNAs which were increased, the mRNAs had to be statistically‘Present’ in the samples which contained minocycline (‘Present’ asdetermined by the Affymetrix microarray suite software). In the case ofmRNAs which were decreased, the mRNAs had to be statistically ‘Present’in the samples which did not contain minocycline.

The three experimental conditions produced three lists of increasedmRNAs, and three lists of decreased mRNAs. The mRNAs common to all threelists are tallied in Table 2, below.

II. Finding mRNAs which are Up-Regulated or Down-Regulated byMinocycline in Samples which are Stimulated with LPS

For both J774.2 and RAW264.7 cells stimulated by LPS, two lists weregenerated, one of mRNAs which were increased at lease 2-fold byminocycline, and one of mRNAs which were decreased at least 2-fold byminocycline. In the case of mRNAs which were increased, the mRNAs had tobe statistically ‘Present’ in the samples which contained minocycline(‘Present’ as determined by the Affymetrix microarray suite software).In the case of mRNAs which were decreased, the mRNAs had to bestatistically ‘Present’ in the samples which did not containminocycline.

The three experimental conditions produced three lists of increasedmRNAs, and three lists of decreased mRNAs. The mRNAs common to all threelists are tallied in Table 2, below.

III. Finding mRNAs which are Up-Regulated or Down Regulated by CompoundsA and B in Samples Also Stimulated with LPS

For J774.2 cells stimulated by LPS, two lists were generated, one ofmRNAs which were increased at lease 2-fold by Compounds A and/or B, andone of mRNAs which were decreased at least 2-fold by Compounds A and/orB. In the case of mRNAs which were increased, the mRNAs had to bestatistically ‘Present’ in the samples which contained Compounds Aand/or B (‘Present’ as determined by the Affymetrix microarray suitesoftware). In the case of mRNAs which were decreased, the mRNAs had tobe statistically ‘Present’ in the samples which did not containCompounds A and/or B. The structures of compounds A and B are shownbeneath Table 3.

mRNAs were found which were up-regulated by both Compounds A and B, andmRNAs were found which were down-regulated by both compounds. Resultsare tallied in Table 3, below.

TABLE 3 Numbers of mRNAs with Numbers of mRNAs with levels significantlyaltered by Numbers of mRNAs with levels significantly altered byminocycline, in the presence levels significantly altered by minocyclineof LPS both Compounds A and B, in (in J774.2 and RAW264.7 (in J774.2 andRAW264.7 the presence of LPS cells) cells) (in J774.2 only) Increased 2128 133 Decreased 4 9 108

Example 4 Modulation of Inducible Nitric Oxide Synthase (iNOS) withMinocycline Materials and Methods

Mouse macrophage J774.2 cells were seeded into 6 well plates asdescribed above, and exposed to either minocycline alone, or incombination with LPS as above (untreated and LPS-alone conditions wereused as controls). Data representing the modulation of iNOS mRNA wasextracted from the Affymetrix data, described above.

Nitrite levels were measured in the supernatants of the samples usingthe Greiss reaction. Briefly, 100 μl singlicates of supernatant wereincubated in the dark for 10 minutes with sulfanilamide solution (1%sulfanilamide in 5% H₂PO₄). Then 50 μl of NED (0.1%N-1-napthylethylenediamine dihydrochloride in water) was added, and thesamples incubated for a further 10 minutes in the dark. Samples wereread in a Wallac Victor V plate reader at 535 nm.

Protein levels were measured by Western analysis. Cells were lysed in 10mM Tris HCl, pH 7.4, 1 mM EDTA, 0.5% SDS, protease inhibitors and DNAse.The antibody used to detect the iNOS protein was an anti iNOS antibodyfrom Transduction laboratories. The results of the experiment are shownin FIG. 1.

Example 5 Mammalian Cytotoxicity Assay

COS-1 and CHO-K1 cell suspensions were prepared, seeded into 96-welltissue culture treated black-walled microtiter plates (densitydetermined by cell line), and incubated overnight at 37° C., in 5% CO₂and approximately 95% humidity. The following day, serial dilutions ofdrug were prepared under sterile conditions and transferred to cellplates. Cell/Drug plates were incubated under the above conditions for24 hours. Following the incubation period, media/drug was aspirated and50 μl of Resazurin (0.042 mg/ml in PBS w/Ca and Mg) was added. Theplates were then incubated under the above conditions for 2 hours andthen in the dark at room temperature for an additional 30 minutes.Fluorescence measurements were taken (excitation 535 nm, emission 590nm). The IC₅₀ (concentration of drug causing 50% growth inhibition) wasthen calculated. The cytotoxicity of both unsubstituted minocycline anddoxycycline were found to be greater than 25 μg/ml. Each of thecompounds tested was found to have an acceptable cytotoxicity.

Example 6 In Vitro Anti-Bacterial Activity Assay

The following assay was used to determine the efficacy of thetetracycline compounds against gram positive (S. aureus RN450) and gramnegative (E. coli ML308 225) bacteria. 2 mg of each compound wasdissolved in 100 μl of DMSO. The solution was then added tocation-adjusted Mueller Hinton broth (CAMHB), which resulted in a finalcompound concentration of 200 μg per ml. The tetracycline compoundsolutions were diluted to 50 μL volumes, with a test compoundconcentration of 0.098 μg/ml. Optical density (OD) determinations weremade from fresh log-phase broth cultures of the test strains. Dilutionswere made to achieve a final cell density of 1×10⁶ CFU/ml. At OD=1, celldensities for different genera were approximately:

E. coli 1 × 10⁹ CFU/ml S. aureus 5 × 10⁸ CFU/ml

50 μl of the cell suspensions were added to each well of microtiterplates. The final cell density was approximately 5×10⁵ CFU/ml. Theseplates were incubated at 35° C. in an ambient air incubator forapproximately 18 hours. The plates were read with a microplate readerand were visually inspected when necessary. The MIC was defined as thelowest concentration of the tetracycline compound that inhibits growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference.

1. A method for treating a subject for a DTMR, comprising: administeringto said subject an effective amount of a tetracycline compound offormula (I):

in which R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety; R³, R¹⁰, R¹¹ and R¹² are each hydrogen, alkyl,alkenyl, alkynyl, substituted carbonyl, or a prodrug moiety; R⁴ isNR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen, or hydrogen;R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy; R⁶ and R^(6′) are each independently hydrogen,methylene, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl,alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a); R⁸ ishydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl, alkynyl,aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl, heterocyclic,thionitroso, or —(CH₂)₀₋₃NR^(8c)C(=E′)ER^(8a); R⁹ is hydrogen, hydroxyl,halogen, thiol, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,arylalkynyl, acyl, aminoalkyl, heterocyclic, thionitroso, or—(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a); R^(7a), R^(7b), R^(7c), R^(7d), R^(7e),R^(7f), R^(8a), R^(8b), R^(8c), R^(8d), R^(8e), R^(8f), R^(9a), R^(9b),R^(9c), R^(9d), and R^(9f) are each independently hydrogen, acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety; R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; E is CR^(8d)R^(8e), S, NR^(8b) or O; E′ is O, NR^(8f), or S;W is CR^(7d)R^(7e), S, NR^(7b) or O; W′ is O, NR^(7f), or S; X isCHC(R¹³Y′Y), C═CR¹³Y, CR⁶R⁶, S, NR⁶, or O; Y′ and Y are eachindependently hydrogen, halogen, hydroxyl, cyano, sulfhydryl, amino,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; Z is CR^(9d)R^(9e); S,NR^(9b) or O; and Z′ is O, S, or NR^(9f), or a pharmaceuticallyacceptable salt, ester, or enantiomer thereof, such that said DTMR istreated.
 2. The method of claim 1, wherein said effective amount iseffective to modulate translation of said subject's RNA.
 3. The methodof claim 1, wherein said effective amount is effective to modulate thehalf-life of said subject's RNA.
 4. The method of claim 1, wherein saideffective amount is effective to affect message translocation.
 5. Themethod of claim 1, wherein said effective amount is effective tomodulate the binding of proteins to said subject's RNA.
 6. The method ofclaim 1, wherein said effective amount is effective to modulate splicingof said subject's RNA.
 7. The method of claim 1, wherein R², R^(2′),R^(4′), and R^(4″) are each independently hydrogen or alkyl; R³, R¹⁰,R¹¹ and R¹² are each hydrogen; R⁴ is NR^(4′)R^(4″); R⁵ is hydrogen; R⁶and R^(6′) are each independently hydrogen; R⁷ is substituted alkenyl,substituted alkynyl, substituted phenyl, substituted or unsubstitutedfuranyl, acyl, or aminoalkyl; R⁸ is hydrogen; R⁹ is hydrogen; and X isCR^(6′)R⁶.
 8. The method of claim 7, wherein R², R^(2′), and R¹² areeach hydrogen, and R^(4′) and R^(4″) are each methyl.
 9. The method ofclaim 8, wherein R⁷ is substituted or unsubstituted furanyl.
 10. Themethod of claim 8, wherein R⁷ is substituted phenyl.
 11. The method ofclaim 10, wherein said substituted phenyl is substituted with one ormore substituents and further wherein said substituents are eachindependently alkyl, alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino, acylamino, amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, aryl or heterocyclic moiety.
 12. The method of claim 8,wherein R⁷ is substituted alkenyl.
 13. The method of claim 8, wherein R⁷is substituted alkynyl.
 14. The method of claim 8, wherein R⁷ isdialkylamino.
 15. The method of claim 8, wherein R⁷ is acyl.
 16. Themethod of claim 1, wherein said tetracycline compound is:

or a pharmaceutically acceptable salt thereof.
 17. The method of claim1, wherein said subject is a mammal.
 18. The method of claim 17, whereinsaid mammal is a human.
 19. The method of claim 1, wherein saidtetracycline compound is:

or a pharmaceutically acceptable salt thereof.
 20. The method of claim1, wherein said tetracycline compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts, esters and enantiomers thereof.